PROJECT SUMMARY. PROJECT 5, M.TAKAKU. Pioneer transcription factors are capable of binding to closed chromatin and inducing its opening to drive cell reprogramming. The mechanisms of pioneer factors? actions remain largely unknown. Using a model of mesenchymal-to-epithelial transition (MET) in human breast cancer cells, we have discovered that a pioneer factor GATA3 can open chromatin only at a subset of its binding sites. Our central hypothesis is that GATA3 requires appropriate chromatin context to achieve gene activation that are essential for GATA3 mediated cell reprogramming. The overall objectives in this grant are to identify chromatin structures and GATA3 co-factors needed for GATA3-driven chromatin opening and cell reprogramming. The central hypothesis will be tested by pursuing two specific aims: (1) Define the specific chromatin structures required for GATA3 driven MET; and (2) Identify the roles of GATA3 co-factors for epithelial cancer cell reprogramming. We will use in vitro and in vivo breast cancer cell systems to address these specific aims. Under the first aim, the doxycycline inducible GATA3 expression system in MDA-MB-231 cells that we have established will be used to identify the roles of nucleosome positioning and linker histones (H1.3 and H1.5) during GATA3-mediated MET. Genomic techniques including ChIP-seq, ATAC-seq, RNA-seq and MNase-seq together with CRISPR-Cas9 genome editing and shRNA knockdown methods will be utilized. For the second aim, the same inducible GATA3 expression system and a GATA3 mutant T47D cell line (established by our group) will be used to identify the roles of chromatin remodeling factors BRG1 and CHD4 in the GATA3-mediated MET and the GATA3 mutant induced epithelial-to-mesenchymal transition (EMT). It is important to understand the roles of GATA3 co- factors in the GATA3 mutant cell context, because GATA3 was recently identified as one of the most frequently mutated genes in breast cancer. Under both aims, mouse xenograft tumor model will be used to identify in vivo roles of chromatin structures and chromatin remodeling factors in tumor growth and metastasis. The proposed research is innovative in two ways: first, it features novel hypotheses that will advance the fields of transcription, chromatin and cancer biology, and secondly, there are multiple innovative technologies that are proposed, most of which the project leader has developed. The proposed research is significant because it is expected to provide a new framework for understanding the functions of transcription factors, chromatin remodeling factors, and chromatin structures in cancer cell reprogramming. Ultimately, such knowledge has the potential to offer development of new therapeutic strategies of breast cancer treatment.